Ubiquitin-binding Site Research Articles

Disruption of the autoinhibited state primes the E3 ligase parkin for activation and catalysis.

The PARK2 gene is mutated in 50% of autosomal recessive juvenile parkinsonism (ARJP) cases. It encodes parkin, an E3 ubiquitin ligase of the RBR family. Parkin exists in an autoinhibited state that is activated by phosphorylation of its N-terminal ubiquitin-like (Ubl) domain and binding of phosphoubiquitin. We describe the 1.8 Å crystal structure of human parkin in its fully inhibited state and identify the key interfaces to maintain parkin inhibition. We identify the phosphoubiquitin-binding interface, provide a model for the phosphoubiquitin–parkin complex and show how phosphorylation of the Ubl domain primes parkin for optimal phosphoubiquitin binding. Furthermore, we demonstrate that the addition of phosphoubiquitin leads to displacement of the Ubl domain through loss of structure, unveiling a ubiquitin-binding site used by the E2∼Ub conjugate, thus leading to active parkin. We find the role of the Ubl domain is to prevent parkin activity in the absence of the phosphorylation signals, and propose a model for parkin inhibition, optimization for phosphoubiquitin recruitment, release of inhibition by the Ubl domain and engagement with an E2∼Ub conjugate. Taken together, this model provides a mechanistic framework for activating parkin.

Understanding the Molecular Basis of MIC‐CAP Syndrome through Structural and Functional Studies of the Deubiquitinase AMSH

AMSH, associated molecule with SH3 domain of signal transducing adaptor molecule, is a deubiquitinase recruited by the endosomal sorting complex required for transport (ESCRT) machinery. This machinery executes the endocytic sorting of cell-surface receptors tagged with Lys63-linked polyubiquitin chains and delivers them to lysosomes for degradation. Recently, it was found that mutations in AMSH lead to microcephaly-capillary malformation (MIC-CAP) syndrome. Since the molecular basis of the MIC-CAP syndrome is not known, we would like to use fission yeast as a model system, which has an AMSH orthologue, Sst2 to gain insight into the disease. I cloned, purified and solved the crystal structure of Sst2 in free and ubiquitin bound forms. Along with kinetic analysis, these studies led to the conclusion that Sst2 is indeed the AMSH orthologue in fission yeast. I then generated and characterized Sst2 mutants carrying the same mutations found in MIC-CAP syndrome in AMSH. The disease mutant Thr319Ile showed a defect in its kcat in diubiquitin cleavage assays indicating a loss of interactions during the catalytic transition state. Interestingly, another disease mutation, R433X, far from both the ubiquitin binding site and the active site, showed a severe defect in kcat, the basis of which is being further investigated. In order to functionally characterize these mutants, I am using a deletion S. pombe strain (ubp4Δ1 ubp5Δ ubp9Δ ubp15Δ sst2Δ) lacking endogenous Sst2, which shows a defect in endocytosis. Complementation assay using plasmids carrying Sst2 and its mutants will be used to study their effects on endocytosis .These studies will provide insights into how mutations in the AMSH gene can cause MIC-CAP Syndrome.

Structural Basis for the Activation and Inhibition of the UCH37 Deubiquitylase

The UCH37 deubiquitylase functions in two large and very different complexes, the 26S proteasome and the INO80 chromatin remodeler. We have performed biochemical characterization and determined crystal structures of UCH37 in complexes with RPN13 and NFRKB, which mediate its recruitment to the proteasome and INO80, respectively. RPN13 and NFRKB make similar contacts to the UCH37 C-terminal domain but quite different contacts to the catalytic UCH domain. RPN13 can activate UCH37 by disrupting dimerization, although physiologically relevant activation likely results from stabilization of a surface competent for ubiquitin binding and modulation of the active-site crossover loop. In contrast, NFRKB inhibits UCH37 by blocking the ubiquitin-binding site and by disrupting the enzyme active site. These findings reveal remarkable commonality in mechanisms of recruitment, yet very different mechanisms of regulating enzyme activity, and provide a foundation for understanding the roles of UCH37 in the unrelated proteasome and INO80 complexes.

Allosteric regulation of deubiquitylase activity through ubiquitination.

Ataxin-3, the protein responsible for spinocerebellar ataxia type-3, is a cysteine protease that specifically cleaves poly-ubiquitin chains and participates in the ubiquitin proteasome pathway. The enzymatic activity resides in the N-terminal Josephin domain. An unusual feature of ataxin-3 is its low enzymatic activity especially for mono-ubiquitinated substrates and short ubiquitin chains. However, specific ubiquitination at lysine 117 in the Josephin domain activates ataxin-3 through an unknown mechanism. Here, we investigate the effects of K117 ubiquitination on the structure and enzymatic activity of the protein. We show that covalently linked ubiquitin rests on the Josephin domain, forming a compact globular moiety and occupying a ubiquitin binding site previously thought to be essential for substrate recognition. In doing so, ubiquitination enhances enzymatic activity by locking the enzyme in an activated state. Our results indicate that ubiquitin functions both as a substrate and as an allosteric regulatory factor. We provide a novel example in which a conformational switch controls the activity of an enzyme that mediates deubiquitination.

A Computational Study of Mono- and Poly-Ubiquitin Recognition by the Proteasome Subunit Rpn10

Accurate and timely protein degradation is a vital cellular process. Protein degradation is performed by the 26S proteasome complex, a large multi-subunit molecular machine that recognizes, binds, and degrades protein substrates covalently bound to poly-ubiquitin tags. The 26S proteasome, which recognizes ubiquitin tags through its subunit Rpn10, has a preference for binding K48-linked tetra-ubiquitin chains. However, Rpn10 and its homologs usually have only one or two ubiquitin binding sites, located on their long and flexible C-terminal arms. Here, to examine the role of Rpn10 in the 26S proteasome preference for tetra-ubiquitin tags, we perform molecular dynamics simulations of Rpn10 bound to mono-ubiquitin and poly-ubiquitin chains. To overcome timescale and sampling limitations, we use a combination of a hybrid model force field (PACE) and temperature replica exchange molecular dynamics (T-REMD) simulations. The performed simulations characterize in detail Rpn10 binding to its ubiquitin partners and identify transient contacts that could stabilize Rpn10 (and 26S proteasome) binding to poly-ubiquitin chains. Our results demonstrate at atomic resolution how the flexible Rpn10 subunit contributes to the recognition of poly-ubiquitin tags.

Convergent Evolution in the Assembly of Polyubiquitin Degradation Signals by the Shigella flexneri IpaH9.8 Ligase

The human pathogen Shigella flexneri subverts host function and defenses by deploying a cohort of effector proteins via a type III secretion system. The IpaH family of 10 such effectors mimics ubiquitin ligases but bears no sequence or structural homology to their eukaryotic counterpoints. Using rates of (125)I-polyubiquitin chain formation as a functional read out, IpaH9.8 displays V-type positive cooperativity with respect to varying concentrations of its Ubc5B∼(125)I-ubiquitin thioester co-substrate in the nanomolar range ([S]½ = 140 ± 32 nm; n = 1.8 ± 0.1) and cooperative substrate inhibition at micromolar concentrations ([S]½ = 740 ± 240 nm; n = 1.7 ± 0.2), requiring ordered binding to two functionally distinct sites per subunit. The isosteric substrate analog Ubc5BC85S-ubiquitin oxyester acts as a competitive inhibitor of wild-type Ubc5B∼(125)I-ubiquitin thioester (Ki = 117 ± 29 nm), whereas a Ubc5BC85A product analog shows noncompetitive inhibition (Ki = 2.2 ± 0.5 μm), consistent with the two-site model. Re-evaluation of a related IpaH3 crystal structure (PDB entry 3CVR) identifies a symmetric dimer consistent with the observed cooperativity. Genetic disruption of the predicted IpaH9.8 dimer interface reduces the solution molecular weight and significantly ablates the kcat but not [S]½ for polyubiquitin chain formation. Other studies demonstrate that cooperativity requires the N-terminal leucine-rich repeat-targeting domain and is transduced through Phe(395). Additionally, these mechanistic features are conserved in a distantly related SspH2 Salmonella enterica ligase. Kinetic parallels between IpaH9.8 and the recently revised mechanism for E6AP/UBE3A (Ronchi, V. P., Klein, J. M., and Haas, A. L. (2013) E6AP/UBE3A ubiquitin ligase harbors two E2∼ubiquitin binding sites. J. Biol. Chem. 288, 10349-10360) suggest convergent evolution of the catalytic mechanisms for prokaryotic and eukaryotic ligases.

Dss1 Is a 26S Proteasome Ubiquitin Receptor

SummaryThe ubiquitin-proteasome system is the major pathway for protein degradation in eukaryotic cells. Proteins to be degraded are conjugated to ubiquitin chains that act as recognition signals for the 26S proteasome. The proteasome subunits Rpn10 and Rpn13 are known to bind ubiquitin, but genetic and biochemical data suggest the existence of at least one other substrate receptor. Here, we show that the phylogenetically conserved proteasome subunit Dss1 (Sem1) binds ubiquitin chains linked by K63 and K48. Atomic resolution data show that Dss1 is disordered and binds ubiquitin by binding sites characterized by acidic and hydrophobic residues. The complementary binding region in ubiquitin is composed of a hydrophobic patch formed by I13, I44, and L69 flanked by two basic regions. Mutations in the ubiquitin-binding site of Dss1 cause growth defects and accumulation of ubiquitylated proteins.

Investigation of the Josephin Domain protein-protein interaction by molecular dynamics.

Spinocerebellar ataxia (SCA) 3, the most common form of SCA, is a neurodegenerative rare disease characterized by polyglutamine tract expansion and self-assembly of Ataxin3 (At3) misfolded proteins into highly organized fibrillar aggregates. The At3 N-terminal Josephin Domain (JD) has been suggested as being responsible for mediating the initial phase of the At3 double-step fibrillogenesis. Several issues concerning the residues involved in the JD’s aggregation and, more generally, the JD clumping mechanism have not been clarified yet. In this paper we present an investigation focusing on the JD protein-protein interaction by means of molecular modeling. Our results suggest possible aminoacids involved in JD contact together with local and non-local effects following JD dimerization. Surprisingly, JD conformational changes following the binding may involve ubiquitin binding sites and hairpin region even though they do not pertain to the JD interaction surfaces. Moreover, the JD binding event has been found to alter the hairpin open-like conformation toward a closed-like arrangement over the simulated timescale. Finally, our results suggest that the JD aggregation might be a multi-step process, with an initial fast JD-JD binding mainly driven by Arg101, followed by slower structural global rearrangements involving the exposure to the solvent of Leu84-Trp87, which might play a role in a second step of JD aggregation.

Abstract 517: Ubiquitylation of G Protein Coupled Receptor Kinase 4 (GRK4) Regulates its Localization and Abundance

The dopamine-1 receptor (D 1 R) is responsible for regulating up to 60% of natriuresis in the kidney under sodium loaded conditions. G protein coupled receptor kinase type 4 (GRK4) phosphorylates the D 1 R and reduces its membrane expression, but little is known about regulation of GRK4. We hypothesized that GRK4 is targeted for elimination through ubiquitylation. Using D 1 R and GRK4γ stably transfected human embryonic kidney cells (HEK293), we demonstrated GRK4 and ubiquitin coimmunoprecipitation. Addition of clasto-lactacystin beta-lactone (CLBL, an inhibitor of the 26S proteasome) increased GRK4 expression levels (western blot) using two different GRK4 fusion protein constructs. The addition of CLBL (10 μmol/L, 24 hrs) increased the expression of both a tandem affinity tagged (tap-tag) GRK4 fusion construct (2.65±0.19 fold over vehicle (VEH): VEH 4,010±404 RFU; CLBL 10,626±744 RFU, P<0.05, N=3) as well as a mCherry GRK4 fusion construct (6.98±0.50 fold increase over VEH: VEH 5,044±3229 RFU; CLBL 35,210±2547 RFU, P<0.05, N=3). The ubiquitin binding sites (AA 216,217; MYACK Ub K Ub LQKK) are located near the nuclear localization signal (AA 219-228; QKKRIKKRK) in GRK4. We therefore examined the subcellular localization of GRK4 after CLBL treatment. Nuclear accumulation of the GRK4-mCherry fusion protein fluorescence decreased markedly while cytoplasmic GRK4-mCherry fluorescence increased (nuclear to cytoplasmic GRK4 ratio: VEH 1.18±0.09 RFU; CLBL 0.78±0.03 RFU, P<0.01, N=12). In summary, GRK4 expression levels are negatively regulated by the ubiquitin proteasome system and ubiquitylation of GRK4 prevents nuclear import. We hypothesize that ubiquitylation is sterically hindering the nuclear localization sequence in GRK4 from interacting with the nuclear import machinery of the cell. Through manipulation of GRK4 ubiquitylation, one may be able to separate nuclear vs. cytoplasmic activity of GRK4 and reverse the negative impact overactive GRK4 has on dopaminergic signaling.

Ubiquitin-binding site 2 of ataxin-3 prevents its proteasomal degradation by interacting with Rad23.

Polyglutamine repeat expansion in ataxin-3 causes neurodegeneration in the most common dominant ataxia, Spinocerebellar Ataxia Type 3 (SCA3). Since reducing levels of disease proteins improves pathology in animals, we investigated how ataxin-3 is degraded. Here we show that, unlike most proteins, ataxin-3 turnover does not require its ubiquitination, but is regulated by Ubiquitin-Binding Site 2 (UbS2) on its N terminus. Mutating UbS2 decreases ataxin-3 protein levels in cultured mammalian cells and in Drosophila melanogaster by increasing its proteasomal turnover. Ataxin-3 interacts with the proteasome-associated proteins Rad23A/B through UbS2. Knockdown of Rad23 in cultured cells and in Drosophila results in lower levels of ataxin-3 protein. Importantly, reducing Rad23 suppresses ataxin-3-dependent degeneration in flies. We present a mechanism for ubiquitination-independent degradation that is impeded by protein interactions with proteasome-associated factors. We conclude that UbS2 is a potential target through which to enhance ataxin-3 degradation for SCA3 therapy.

Novel ubiquitin binding sites of S. cerevisiae polymerase η identified by a genetically encoded photoactive probe (927.4)

DNA translesion synthesis is carried out by specialized DNA polymerases such as Polη. Polη contains a ubiquitin‐binding zinc finger (UBZ) domain required for its effective recruitment to the stalled replication fork in response to DNA damages. Our previous study reported a novel ubiquitin‐binding mode of S. cerevisiae Polη in which the C‐terminal portion of Polη binds ubiquitin with 30‐fold higher affinity than the UBZ domain alone. However, the mechanism of such higher binding affinity is not clear. In this study, a genetically encoded photoactive probe, p‐benzoyl‐L‐phenylalanine (pBpa), was incorporated at various positions on ubiquitin to map the interaction sites between Polη and ubiquitin. Data suggests that besides the Ile44 hydrophobic patch which was known to bind UBZ domain of Polη, several other residues at the ubiquitin C‐terminus and a surface opposite from the hydrophobic patch on ubiquitin also bind to Polη. These new binding sites increase the binding affinity between ubiquitin and Polη. Our finding is important for understanding the role of ubiquitin in the recruitment of Polη during DNA translesion synthesis.Grant Funding Source: Supported by U.S National Science Foundation Grant MCB‐0953764 to Z. Zhuang

Ubiquitin code assembly and disassembly

Ubiquitin, a 76 amino-acid polypeptide, presents a compact three-dimensional structure, utilising a fold that recurs within larger polypeptides and in other protein modifiers, such as NEDD8 and SUMO. Ubiquitylation was initially recognised as a signal for proteasome-mediated degradation. We shall consider here how this view has evolved to appreciate that the dynamic appendage of different types of ubiquitin chains represents a versatile, three-dimensional code, fundamental to the control of many cellular processes.

E2 enzyme inhibition by stabilization of a low-affinity interface with ubiquitin.

Weak protein interactions between ubiquitin and the ubiquitin-proteasome system (UPS) enzymes that mediate its covalent attachment to substrates serve to position ubiquitin for optimal catalytic transfer. We show that a small molecule inhibitor of the E2 ubiquitin conjugating enzyme Cdc34A, called CC0651, acts by trapping a weak interaction between ubiquitin and the E2 donor ubiquitin binding site. A structure of the ternary CC0651-Cdc34A-ubiquitin complex reveals that the inhibitor engages a composite binding pocket formed from Cdc34A and ubiquitin. CC0651 also suppresses the spontaneous hydrolysis rate of the Cdc34A-ubiquitin thioester, without overtly affecting the interaction between Cdc34A and the RING domain subunit of the E3 enzyme. Stabilization of the numerous other weak interactions between ubiquitin and UPS enzymes by small molecules may be a feasible strategy to selectively inhibit different UPS activities.

THU0026 P62/SQSTM1 links reactive oxygen species formation and obesity to increased tnfalpha-mediated joint destruction via its signalling domains

BackgroundThe multidomain protein p62/SQSTM1 modulates important signalling pathways responsible for osteoclastogenesis, cell proliferation and polarity. Furthermore, it also regulates protein degradation by autophagy, the ubiquitin proteasome system or sequestration. Although...

Novel Inhibitors of Rad6 Ubiquitin Conjugating Enzyme: Design, Synthesis, Identification, and Functional Characterization

Protein ubiquitination is important for cell signaling, DNA repair, and proteasomal degradation, and it is not surprising that alterations in ubiquitination occur frequently in cancer. Ubiquitin-conjugating enzymes (E2) mediate ubiquitination by selective interactions with ubiquitin-activating (E1) and ubiquitin ligase (E3) enzymes, and thus selective E2 small molecule inhibitor (SMI) will provide specificity unattainable with proteasome inhibitors. Here we describe synthesis and functional characterization of the first SMIs of human E2 Rad6B, a fundamental component of translesion synthesis DNA repair. A pharmacophore model for consensus E2 ubiquitin-binding sites was generated for virtual screening to identify E2 inhibitor candidates. Twelve triazine (TZ) analogs screened in silico by molecular docking to the Rad6B X-ray structure were verified by their effect on Rad6B ubiquitination of histone H2A. TZs #8 and 9 docked to the Rad6B catalytic site with highest complementarity. TZs #1, 2, 8, and 9 inhibited Rad6B-ubiquitin thioester formation and subsequent ubiquitin transfer to histone H2A. SMI #9 inhibition of Rad6 was selective as BCA2 ubiquitination by E2 UbcH5 was unaffected by SMI #9. SMI #9 more potently inhibited proliferation, colony formation, and migration than SMI #8, and induced MDA-MB-231 breast cancer cell G2-M arrest and apoptosis. Ubiquitination assays using Rad6 immunoprecipitated from SMI #8- or 9-treated cells confirmed inhibition of endogenous Rad6 activity. Consistent with our previous data showing Rad6B-mediated polyubiquitination stabilizes β-catenin, MDA-MB-231 treatment with SMIs #8 or 9 decreased β-catenin protein levels. Together these results describe identification of the first Rad6 SMIs.

RING-between-RINGs-keeping the safety on loaded guns

The ‘RING‐between‐RING’‐type E3 ubiquitin ligase HOIP acts via a novel RING/HECT‐hybrid ubiquitin transfer mechanism and catalyses the formation of linear ubiquitin chains by non‐covalently binding the acceptor ubiquitin. But in the absence of a binding partner, HOIP is auto‐inhibited. This explains why assembly of either HOIP/HOIL‐1L or HOIP/SHARPIN is required to catalyse linear chain formation.

Specific recognition of linear polyubiquitin by A20 zinc finger 7 is involved in NF-κB regulation

LUBAC (linear ubiquitin chain assembly complex) activates the canonical NF-κB pathway through linear polyubiquitination of NEMO (NF-κB essential modulator, also known as IKKγ) and RIP1. However, the regulatory mechanism of LUBAC-mediated NF-κB activation remains elusive. Here, we show that A20 suppresses LUBAC-mediated NF-κB activation by binding linear polyubiquitin via the C-terminal seventh zinc finger (ZF7), whereas CYLD suppresses it through deubiquitinase (DUB) activity. We determined the crystal structures of A20 ZF7 in complex with linear diubiquitin at 1.70-1.98 Å resolutions. The crystal structures revealed that A20 ZF7 simultaneously recognizes the Met1-linked proximal and distal ubiquitins, and that genetic mutations associated with B cell lymphomas map to the ubiquitin-binding sites. Our functional analysis indicated that the binding of A20 ZF7 to linear polyubiquitin contributes to the recruitment of A20 into a TNF receptor (TNFR) signalling complex containing LUBAC and IκB kinase (IKK), which results in NF-κB suppression. These findings provide new insight into the regulation of immune and inflammatory responses.

OTUB1 Co-opts Lys48-Linked Ubiquitin Recognition to Suppress E2 Enzyme Function

Ubiquitylation entails the concerted action of E1, E2, and E3 enzymes. We recently reported that OTUB1, a deubiquitylase, inhibits the DNA damage response independently of its isopeptidase activity. OTUB1 does so by blocking ubiquitin transfer by UBC13, the cognate E2 enzyme for RNF168. OTUB1 also inhibits E2s of the UBE2D and UBE2E families. Herewe elucidate the structural mechanism by which OTUB1 binds E2s to inhibit ubiquitin transfer. OTUB1 recognizes ubiquitin-charged E2s through contacts with both donor ubiquitin and the E2 enzyme. Surprisingly, free ubiquitin associates with the canonical distal ubiquitin-binding site on OTUB1 to promote formation of the inhibited E2 complex. Lys48 of donor ubiquitin lies near the OTUB1 catalytic site and the C terminus of free ubiquitin, a configuration that mimics the products of Lys48-linked ubiquitin chain cleavage. OTUB1 therefore co-opts Lys48-linked ubiquitin chain recognition to suppress ubiquitin conjugation and the DNA damage response.

An ankyrin-repeat ubiquitin-binding domain determines TRABID's specificity for atypical ubiquitin chains

Eight different types of ubiquitin (Ub) linkages are present in eukaryotic cells that regulate diverse biological processes. Proteins that mediate specific assembly and disassembly of atypical Lys6, Lys27, Lys29 and Lys33 linkages are largely unknown. We here reveal how the human Ovarian Tumor (OTU) domain deubiquitinase (DUB) TRABID specifically hydrolyzes both Lys29- and Lys33-linked diubiquitin (diUb). A crystal structure of the extended catalytic domain reveals an unpredicted Ankyrin repeat (Ank) domain that precedes an A20-like catalytic core. NMR analysis identifies the Ank domain as a new Ub binding fold termed AnkUBD, and DUB assays in vitro and in vivo show that this domain is crucial for TRABID efficiency and linkage-specificity. Our data are consistent with a role of the AnkUBD as an enzymatic S1' Ub binding site, which orients a Ub chain such that Lys29 and Lys33 linkages are cleaved preferentially.

Fragmentation methods on the balance: unambiguous top–down mass spectrometric characterization of oxaliplatin–ubiquitin binding sites

The interaction between oxaliplatin and the model protein ubiquitin (Ub) was investigated in a top-down approach by means of high-resolution electrospray ionization mass spectrometry (ESI-MS) using diverse tandem mass spectrometric (MS/MS) techniques, including collision-induced dissociation (CID), higher-energy C-trap dissociation (HCD), and electron transfer dissociation (ETD). To the best of our knowledge, this is the first time that metallodrug-protein adducts were analyzed for the metal-binding site by ETD-MS/MS, which outperformed both CID and HCD in terms of number of identified metallated peptide fragments in the mass spectra and the localization of the binding sites. Only ETD allowed the simultaneous and exact determination of Met1 and His68 residues as binding partners for oxaliplatin. CID-MS/MS experiments were carried out on orbitrap and ion cyclotron resonance (ICR)-FT mass spectrometers and both instruments yielded similar results with respect to number of metallated fragments and the localization of the binding sites. A comparison of the protein secondary structure with the intensities of peptide fragments generated by collisional activation of the [Ub+Pt-(chxn)] adduct [chxn = (1R,2R)-cyclohexanediamine] revealed a correlation with cleavages in solution phase random coil areas, indicating that the N-terminal β-hairpin and α-helix structures are retained in the gas phase.